TREA

Eukaryotic regulatable transcription

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Information

  • Patent Grant
  • 4880734
  • Patent Number
    4,880,734
  • Date Filed
    Thursday, May 29, 1986
    38 years ago
  • Date Issued
    Tuesday, November 14, 1989
    34 years ago
Information
Abstract
Methods and compositions are provided for efficient expression of genes in unicellular microoorganisms, particularly yeast. The systems involve an expression system employing transcriptional initiation regions from glycolytic enzymes, particularly a chimeric expression system, having a first region providing for regulatable or constitutive expression, a second region providing for transcriptional initiation, where regions one and two are not found joined together in functional relationship in nature, and optionally a sequence providing for a secretory leader and processing signal, where the expression cassette will be joined to a gene which may be homologous or heterologous to the host. The expression cassette can be used on an extrachromosomal element or integrated into the host genome, whereby continuous expression can be achieved or inducible expression is obtained, by virtue of the presence or absence of an inducer. Constructions may be provided, where structural genes may be introduced in reading frame with the cassette to provide for expression of the natural peptide, a fused peptide, or a peptide precursor.The S. cerevisiae strain AB110 (pAB24/P. vivax 1-5 was deposited at the A.T.C.C. on May 9, 1986, and given Accession No. 20797.
Description
Claims
  • 1. A DNA construct comprising in order of transcription a first transcriptional regulatory region obtained from one of the yeast genes ADR3, GAL4, or the regulatory region of PHO5, which provides for inducible transcriptional regulation; a second transcriptional initiation region from the yeast GAPDH gene of FIG. 2; a structural gene coding for other than the wild-type gene of said transcriptional initiation region; and a terminator region.
  • 2. A DNA construct according to claim 1, wherein induction results from a change in concentration of an organic or inorganic nutrient.
  • 3. A DNA construct according to claim 1, wherein induction results from a change in temperature.
  • 4. An expression vector comprising:
  • a yeast replication system;
  • and an expression cassette comprising in order of transcription a first transcriptional regulatory region obtained from one of the yeast genes ADR3, GAL4, or the regulatory region of PHO5, which provides for inducible transcriptional regulation in yeast;
  • a transcriptional initiation region from the yeast GAPDH gene of FIG. 2;
  • a structural gene coding for other than the wild-type gene of said transcriptional initiation region; and
  • a terminator region.
  • 5. An expression vector according to claim 4, wherein said first transcriptional regulatory region is inducible by changing concentration of an organic or inorganic nutrient.
  • 6. An expression vector according to claim 4, wherein said first transcriptional regulatory region is inducible by a temperature change.
  • 7. An expression vector according to claim 4, wherein said structural gene comprises the .alpha.-secretory leader, processing signal and an open reading frame coding for a polypeptide of at least about 12 amino acids.
  • 8. A yeast host having an episomal element according to claim 4.
  • 9. A method for producing a polypeptide which comprises growing a yeast host according to claim 8 in an appropriate nutrient medium;
  • whereby the structural gene is expressed to produce said peptide.
  • 10. A method according to claim 9, wherein said structural gene includes the .alpha.-factor leader and processing signal, whereby said peptide is processed and secreted.
  • 11. A method according to claim 9, wherein said peptide is retained in the cytoplasm of said yeast host, and including the additional steps of isolating said peptide from said yeast host.
  • 12. A DNA construct comprising in order of transcription a first transcriptional regulatory region obtained from one of the yeast genes ADR3, GAL4, or the regulatory region of PHO5; a second transcriptional initiation region from the yeast GAPDH gene of FIG. 2; a structural gene coding for other than the wild-type gene of said transcriptional initiation region; and a terminator region, wherein said DNA construct is a linear fragment free of other DNA or joined to other DNA functional in a cellular host.
  • 13. A DNA construct according to claim 12, wherein said other DNA includes a replication system functional in a prokaryotic host and/or yeast.
  • 14. A DNA construct according to any of claims 12 or 13, wherein said structural gene includes at its 5' terminus .alpha.-factor leader sequence and processing signal.
  • 15. A DNA construct according to claim 12, wherein said structural gene codes for at least an immunologically active fragment of a malarial circumsporozoite protein.
  • 16. A DNA construct according to claim 12, wherein said structural gene codes for at least an immunologically active fragment of superoxide dismutase.
  • 17. A DNA construct according to claim 12, wherein said structural gene codes for at least an immunologically active fragment of .alpha.1-antitrypsin or mutants thereof having enzyme inhibitory activity.
  • 18. A DNA construct according to claim 12, wherein said structural gene codes for at least an immunologically active fragment of hepatitis B surface antigen.
  • 19. A DNA construct according to claim 12, wherein said structural gene codes for at least an immunologically active fragment of insulin.
  • 20. A DNA construct according to claim 12, wherein said structural gene codes for an SOD-insulin fusion product.
  • 21. A transcriptional regulation control region comprising in the direction transcription:
  • a transcriptional regulatory region obtained from one of the yeast genes GAL4, ADR3, or the regulatory region of PHO5; and
  • a yeast transcription initiation region from the yeast glycolytic enzyme GAPDH shown in FIG. 2, wherein said transcriptional initiation region is joined through an untranslated region to a sequence encoding for alphafactor leader peptide and a processing signal joined to a structural gene in reading frame; and
  • a DNA sequence functional in a yeast host, other than the wild-type gene of said transcription initiation region.
CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. application Ser. No. 760,197 filed July 29, 1985, which is a continuation-in-part of U.S. application Ser. No. 468,589 filed Feb. 22, 1983, now abandoned, and U.S. application Ser. No. 609,540 filed May 11, 1984, now abandoned, which disclosures are incorporated herein by reference. 1. Field of the Invention The advent of hybrid DNA technology provided a unique opportunity to produce peptides of any sequence of naturally-occurring amino acids. Thus, for the first time, macromolecules could be prepared at will from a large number of different monomers to provide a specifically defined sequence. Once the feasibility had been established, there was then an interest in the economics of the technology, in providing systems which could be adapted to the production of particular products. In part, because of the familiarity with E. coli, E. coli was an obvious host for research expression and potentially for commercial expression of products of interest. However, E. coli, as well as other prokaryotes, have a number of disadvantages. Many of the prokaryotes produce endo- or exotoxins. Therefore, the resulting product must be carefully purified to ensure the absence of any materials which would affect the health of the patient. This can be particularly troublesome, where the product is administered chronically. Yeast as a host does not suffer from many of the disadvantages of prokaryotes and, furthermore, has a number of advantages. Yeast has been used for a long time in fermentation, so that there are a number of commercial hosts which have a number of desirable properties, such as resistance to viral infection, rapid growth, stability, and the like. In addition, yeast can provide in certain situations glycosylation of the product, so as to provide a peptide product which has a verisimilitude to the naturally-occurring glycosylated product. In order to use yeast as a host, it will be necessary to provide a number of different constructs which allow for expression under a variety of conditions. In many situations, one may wish to have controlled expression, where a change in the nutrient medium may actuate or inhibit expression. Furthermore, since yeast secretes a number of different products naturally, the secretion mechanism may be available for secretion of a variety of peptides foreign to yeast. Thus, yeast provide an attractive opportunity for the development of economical and efficient production of peptides. 2. Description of the Prior Art Krebs, J. Biol. Chem. (1953) 200: 471 and Maitra and Lobo, ibid. (1971) 246: 475 describe properties of glyceraldehyde-3-phosphate dehydrogenate (GAPDH) in yeast. Cloning of GAPDH genes or the pyruvate kinase (PyK) gene has been described by Holland, et al., Basic Life Science (1981) 19: 291; and Kawasaki and Fraenkel, Biochem. Biophys. Res. Comm. (1982) 108: 1107. Yeast promoters which have been linked to foreign genes include alcohol dehydrogenase I (ADHI) (Valenzuela, et al., Nature (1982) 298: 347; Hitzeman, et al., ibid. (1981) 293: 717) and phosphoglycerate kinase (Tuite, et al., EMBO (1980) 1: 603; Hitzeman, et al., Science (1983) 219: 620). Beier and Young, Nature (1982) 300: 724 reports the use of ADR3 as a regulatory sequence. GB 2,154,240 published Sept. 4, 1985, describes the immunodominant epitope of the circumsporozoite protein. See also, Nussenzweig and Nussenzweig, Cell ( 1985) 42: 401-403 and Arnot et al., Science (1985) 230: 815-817, which describe the gene for the immunodominant protein. Novel DNA constructs are provided including a transcription control region comprising a first transcriptional regulatory region and a second transcriptional initiation region, where the two regions may be derived from different sources. Particularly, the transcriptional initiation regions are associated with yeast glycolytic enzymes, while the transcriptional regulatory regions may or may not be derived from regions associated with yeast glycolytic enzymes. The transcriptional control region (the regulatory region and initiation region) is joined to a gene not naturally associated with the transcriptional control region. A terminator region is also present linked to the foreign gene to assure transcription termination. In conjunction, these elements provide for an expression construct ("expression cassette") which can be introduced into a yeast host for maintenance as an extrachromosomal element or integration into the yeast genome. Optionally, a secretory leader can be provided which allows for secretion of the desired peptide product or for targeting of the product onto the secretion pathway.

US Referenced Citations (2)
Number Name Date Kind
4599311 Kawasaki Jul 1986
4615974 Kingsman Oct 1985
Foreign Referenced Citations (2)
Number Date Country
0073657 Mar 1983 EPX
2154240 Sep 1985 GBX
Non-Patent Literature Citations (23)
Entry
Kramer et al. (1984) PNAS 81: 367-370.
Guarente et al. (1981) PNAS 79: 7401-7414.
Tuite et al. (1982) EMBOJ: 603-608.
V. Nussenzweig et al., Cell (1985) 42:401-03.
D. E. Arnot et al., Science (1985) 230: 815-18.
R. A. Kramer et al., Proc Nat Acad Sci U.S.A., (1984) 81: 367-70.
R. A. Hitzeman et al., Nuc Acids Res (1983) 11:2745-63.
A. Miyanohara et al., Proc Nat. Acad Sci U.S.A., (Jan., 1983) 80: 1-5.
L. Guarente et al., Proc Nat Acad Sci U.S.A., (Dec., 1982) 79: 7410-14.
G. Kawasaki et al., Biochem & Biophys Res Comm (1982) 108: 1107-12 (Kawasaki II).
B. Meyhack et al., EMBO J (1982) 1: 675-80.
P. Valenzuela et al., Nature (1982) 298: 347-50.
M. F. Tuite et al, EMBO J (1982) 1: 603-08.
D. R. Beier et al., Nature (Dec., 1982) 300: 724-28.
M. J. Dobson et al., Nuc Acids Res (1982) 10:2625-37.
K. Lerch et al., J Biol Chem (1981) 256: 11545-51.
R. A. Hitzeman et al., Nature (1981) 293: 717-22 (Hitzeman II).
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Ptashne, Nature (1988) 335: 683-689.
Related Publications (1)
Number Date Country
609540 May 1984
Continuation in Parts (2)
Number Date Country
Parent 760197 Jul 1985
Parent 468589 Feb 1983